Rice event GSX2-55

Abstract

The present disclosure provides a rice event GSX2-55 and a detecting and using method thereof, and provides a plant, a plant cell, a tissue or a seed thereof containing a specificity polynucleotide of the event GSX2-55. The present disclosure further provides a measuring method and kit for detecting existence of the rice event GSX2-55 based on DNA sequence of the recombinant construct inserted into rice genome and Flanking sequence of insertion site and a method for generating rice recessive cell nucleus male sterile line.

Claims

1. A recombinant DNA molecule comprising: a) a heterologous nucleic acid molecule inserted into rice genomic DNA and flanking rice genomic sequences thereof, and junction regions of the heterologous nucleic acid molecule to the 5′-end flanking sequence and 3′-end flanking sequence of the rice genome, wherein the heterologous nucleic acid molecule, the flanking sequences thereof and the junction regions are as shown in SEQ ID NO: 10, wherein partial sequence of SEQ ID NO: 10 is SEQ ID NOs: 1-9; the junction region at the 5′ end is as shown in SEQ ID NO: 1, 2, 3, or 7, and the junction region at the 3′ end is as shown in the sequence of 4, 5, 6, or 8; or b) sequences complementary to (a).

2. The recombinant DNA molecule of claim 1, wherein the heterologous nucleic acid molecule comprises DsRed/ZmAA/OsNP1 gene, and has a sequence as shown in SEQ ID NO: 9.

3. The recombinant DNA molecule according to claim 1, wherein the recombinant DNA molecule is derived from transgenic rice plants containing event GSX2-55.

4. The recombinant DNA molecule according to claim 1, wherein the recombinant DNA molecule is comprised in rice plants, plant cells, seeds, and plant parts.

5. The recombinant DNA molecule according to claim 4, wherein the rice seeds produce red fluorescence when excited.

6. The recombinant DNA molecule according to claim 2, wherein the recombinant DNA molecules are amplicons generated from template molecules of event GSX2-55.

7. A DNA probe or primer pair for identifying the presence of event GSX2-55 in a biological sample, wherein the DNA probe or primer pair is designed according to the sequence of SEQ ID NO: 10 and used to identify the presence of the nucleotide sequences of SEQ ID NOs: 1-10 or complementary sequences thereof.

8. The DNA probe or primer pair according to claim 7, wherein the probe or primer has at least 10 nucleotides in length.

9. The DNA probe or primer pair according to claim 8, wherein the probe or primer has at least 18 nucleotides in length.

10. The DNA probe or primer pair according to claim 9, wherein the probe or primer has at least 24 nucleotides in length.

11. A DNA detection kit, characterized by comprising the DNA probe or primer pair for identifying the presence of event GSX2-55 in a biological sample of claim 7.

12. A method for detecting presence of DNA molecule of event GSX2-55 in a biological sample, characterized by comprising the following steps: a) extracting DNA samples from a biological sample; b) detecting the DNA samples by using the DNA probe or primer pair according to claim 7; and c) determining that the event GSX2-55 or progeny thereof is present in the biological sample, if the presence of a nucleotide sequence comprising any one of SEQ ID NOs: 1-10 is detected.

13. A method for producing rice seeds, characterized by comprising the following steps: a) planting rice seeds containing event GSX2-55 in the field, the DNA molecule of event GSX2-55 is the recombinant DNA molecule of claim 1; b) performing selfing of rice comprising the event GSX2-55, or performing sexual crossing of rice containing the event GSX2-55 as a male and second rice plants; c) harvesting rice inbred or hybrid seeds; and d) selecting progeny plants with non-red fluorescent seeds in the excited state, wherein the second rice plants contain osnp1/osnp1 gene that is mutated and loses fertility.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 is a formation of transgenic insertion fragment in a genome of a rice plant containing an event GSX2-55, herein [A] corresponds to a relative position of SEQ ID NO: 1; [B] corresponds to a relative position of SEQ ID NO: 2; [C] corresponds to a relative position of SEQ ID NO: 3; [D] corresponds to a relative position of SEQ ID NO: 4; [E] corresponds to a relative position of SEQ ID NO: 5; [F] corresponds to a relative position of SEQ ID NO: 6; [G] corresponds to a relative position of SEQ ID NO: 7; [H] corresponds to a relative position of SEQ ID NO: 8; [I] corresponds to a relative position of SEQ ID NO: 9; [J] corresponds to a relative position of SEQ ID NO: 10; [K] corresponds to a relative position of SEQ ID NO: 11; [L] corresponds to a relative position of SEQ ID NO: 12; [M] corresponds to a relative position of SEQ ID NO: 13; [N] corresponds to a relative position of SEQ ID NO: 14; [O] corresponds to a relative position (DsRed gene expression cassette) of SEQ ID NO: 19; [P] corresponds to a relative position (ZmAA gene expression cassette) of SEQ ID NO: 20; and [Q] corresponds to a relative position (OsNP1 gene expression cassette) of SEQ ID NO: 21.

[0054] FIG. 2 is sensitivity of detecting the rice event GSX2-55 by quantitative PCR.

[0055] FIG. 3 is contrast of the rice event GSX2-55 seed in a white light state and an excited state respectively, herein the rice event GSX2-55 seed shows the red fluorescence in the excited state.

[0056] FIG. 4 is the rice event GSX2-55 (left) and contrast Huanghuazhan (right) pollen potassium iodide dyeing.

[0057] FIG. 5 is a schematic diagram of producing a rice recessive nucleus male sterile line and hybrid rice by the rice event GSX2-55.

BRIEF DESCRIPTION OF SEQUENCES

[0058] SEQ ID NO: 1 is a sequence of 20 nucleotides, and it shows a5′ connection region of the rice genomic DNA and the integrated transgenic expression cassette. The SEQ ID NO: 1 is positioned in 957-976 bp of a nucleotide position in SEQ ID NO: 10.

[0059] SEQ ID NO: 2 is a sequence of 60 nucleotides, and it shows a 5′ connection region of the rice genomic DNA and the integrated transgenic expression cassette. The SEQ ID NO: 2 is positioned in 937-996 bp of a nucleotide position in the SEQ ID NO: 10.

[0060] SEQ ID NO: 3 is a sequence of 100 nucleotides, and it shows a 5′ connection region of the rice genomic DNA and the integrated transgenic expression cassette. The SEQ ID NO: 3 is positioned in 917-1016 bp of a nucleotide position in the SEQ ID NO: 10.

[0061] SEQ ID NO: 4 is a sequence of 20 nucleotides, and it shows a 3′ connection region of the rice genomic DNA and the integrated transgenic expression cassette. The SEQ ID NO: 4 is positioned in 13555-13574 bp of a nucleotide position in the SEQ ID NO: 10.

[0062] SEQ ID NO: 5 is a sequence of 60 nucleotides, and it shows a 3′ connection region of the rice genomic DNA and the integrated transgenic expression cassette. The SEQ ID NO: 5 is positioned in 13535-13594 bp of a nucleotide position in the SEQ ID NO: 10.

[0063] SEQ ID NO: 6 is a sequence of 100 nucleotides, and it shows a 3′ connection region of the rice genomic DNA and the integrated transgenic expression cassette. The SEQ ID NO: 6 is positioned in 13515-13614 bp of a nucleotide position in the SEQ ID NO: 10.

[0064] SEQ ID NO: 7 is a sequence of 1386 nucleotides, and it shows a 5′ connection region of the rice genomic DNA and the integrated transgenic expression cassette. The SEQ ID NO: 7 is positioned in 1-1386 bp of a nucleotide position in the SEQ ID NO: 10.

[0065] SEQ ID NO: 8 is a sequence of 1440 nucleotides, and it shows a 3′ connection region of the rice genomic DNA and the integrated transgenic expression cassette. The SEQ ID NO: 8 is positioned in 13002-14441 bp of a nucleotide position in the SEQ ID NO: 10.

[0066] SEQ ID NO: 9 is a sequence of 12598 nucleotides, and it shows an integrated transgenic expression cassette sequence. The SEQ ID NO: 9 is positioned in 967-13564 bp of a nucleotide position in the SEQ ID NO: 10.

[0067] SEQ ID NO: 10 is a continuous nucleotide sequence of a 5′ sequence of a side-adjacent inserted DNA, the integrated transgenic expression cassette sequence and a 3′ sequence of the side-adjacent inserted DNA, and the SEQ ID NO: 10 contains the SEQ ID NO: 1-9.

[0068] SEQ ID NO: 11 is a nucleotide sequence of primer TTY-55F of the rice event GSX2-55 identified by using TAQMAN® method, and is complementary with an expression cassette sequence inserted at the 3′ terminal. A primer combination TTY-55F and TTY-55R (SEQ ID NO: 13) is amplified in TAQMAN® (Applied Biosystems) so as to generate a PCR amplicon and diagnose the rice event GSX2-55.

[0069] SEQ ID NO: 12 is a nucleotide sequence of probe TP55 of the rice event GSX2-55 identified by using TAQMAN® method, and it is complementary with a sequence of a 3′ terminal junction region, the 5′ terminal of probe TP55 performs 6-FAM™ labelling, and 3′ terminal performs Minor Groove Binder (MGB) labelling, a primer combination TTY-55F and TTY-55R (SEQ ID NO: 11 and SEQ ID NO: 13), in an amplification reaction combined with the TP55 probe, releases a fluorescence signal and diagnoses the rice event GSX2-55.

[0070] SEQ ID NO: 13 is a nucleotide sequence of primer TTY-55R of the rice event GSX2-55 identified by using TAQMAN® method, and is complementary with a rice genome sequence of the 3′ terminal. A primer combination TTY-55F (SEQ ID NO: 11) and TTY-55R is amplified in the TAQMAN® (Applied Biosystems) so as to generate the PCR amplicon and diagnose the rice event GSX2-55.

[0071] SEQ ID NO: 14 is a nucleotide sequence of the PCR amplicon generated by amplifying the combination of the primers TTY-55F and TTY-55R from the TAQMAN® (Applied Biosystems).

[0072] SEQ ID NO: 15 is a nucleotide sequence of primer TSPS-F of the rice endogenous SPS (Sucrose Phosphate Synthase) gene identified by using the TAQMAN® method. A combination of the primer combination TSPS-F and TSPS-R (SEQ ID NO: 17) is amplified from the TAQMAN® (Applied Biosystems) so as to generate the PCR amplicon, and identity whether the rice genome sequence is existent.

[0073] SEQ ID NO: 16 is a nucleotide sequence of probe TPSPS of the rice endogenous SPS gene identified by using the TAQMAN® method, the 5′ terminal of the probe TPSPS performs VIC™ labelling, and 3′ terminal performs Minor Groove Binder (MGB) labeling, in the amplification reaction of using TSPS-F and TSPS-R (SEQ ID NO: 15 and SEQ ID NO: 17) to be combined with the probe TP55, the fluorescence signal is released and it is diagnosed whether the rice genome sequence is existent.

[0074] SEQ ID NO: 17 is a nucleotide sequence of primer TSPS-R of the rice endogenous SPS gene identified by using the TAQMAN® method. A combination of the primers TSPS-F and TSPS-R is amplified from the TAQMAN® (Applied Biosystems) so as to generate the PCR amplicon, and identity whether the rice genome sequence is existent.

[0075] SEQ ID NO: 18 is a nucleotide sequence of the PCR amplicon generated by amplifying the combination of the primers TSPS-F and TSPS-R (SEQ ID NO: 15 and SEQ ID NO: 17) from the TAQMAN® (Applied Biosystems).

[0076] SEQ ID NO: 19 is the DsRed gene expression cassette nucleotide sequence.

[0077] SEQ ID NO: 20 is the ZmAA gene expression cassette nucleotide sequence.

[0078] SEQ ID NO: 21 is the OsNP1 gene expression cassette nucleotide sequence.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0079] Unless otherwise specified, methods used in the following embodiments are common methods recorded in molecular biology, tissue culture technology and agricultural handbook. For example, the specific steps may see: Molecular Cloning: A Laboratory Manual (3rd edition) (Sambrook, J., Russell, David W., 2001, Cold Spring Harbor), Plant Propagation by Tissue Culture (Edwin F. George, Michael A. Hall, Geert-Jan DeKlerk, 2008, Springer).

[0080] The following embodiments are contained to illuminate instances of some preferable implementation modes of the present disclosure. Those skilled in the art should recognize that technologies disclosed in the following embodiments represent a mode that the inventor discovers the instances which is expressed well in the embodiments of the present disclosure and thereby regarded as the preferable implementation modes thereof. However, according to the present disclosure, those skilled in the art should recognize that many variations may be made in the disclosed specific implementation mode and analogous or similar results are still acquired without departing from spirit and scope of the present disclosure.

Embodiment 1: Rice Transformation and Selection of the Rice Event GSX2-55

[0081] The embodiment describes generation, analysis and selection of the rice event GSX2-55. Specifically, agrobacterium-mediated method is used for performing co-transformation on rice (Hiei et al., Efficient transformation of rice mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. (1994) Plant J6(2):271-282, it is incorporated into the text by reference), Zhen18A is used as a transformation acceptor material, and agrobacterium is used for importing DsRed (SEQ ID NO: 19), ZmAA (SEQ ID NO: 20) and OsNP1 gene (SEQ ID NO: 21) nucleotide sequences into the Zhen18A (Chang et al., Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene (2016) PNAS. 113(49):14145-14150.Fig.S7), as to obtain 1000 strains positive transgenic material or more. After that, through copy number analysis, vector backbone analysis and insertion site analysis, and agronomic traits (including seed setting rate, seed fluorescence segregation ratio, pollen inactivation rate, plant height, growing period and the like), the rice event GSX2-55 is preferably selected therefrom.

Embodiment 2: Pollen Fertility Detection of the Rice Event GSX2-55

[0082] Pollen activity observation analysis is performed on the rice event GSX2-55 and non-transgenic controlled rice (Hemeizhan and Huanghuazhan) in the embodiment 1. Specifically, in a later blooming period of the rice, a single plant is randomly extracted from the above three varieties, one adequate mature flower bud to be bloomed is taken from each plant, and an anther is taken from each flower, the anther is placed on a glass slide, and a drop of distilled water is added, after pollen grains are released by tweezers, 1-2 drops of 1.sub.2-KI (enabling 2 g of KI and 1 g of 1.sub.2 to be dissolved in 300 ml of the distilled water) solution is added, covered by cover glass, and observed under a low-magnification microscope, the number of dyeing pollens and the number of non-dyeing pollens are counted. If the pollen grains are blue, it is indicated that the pollen grains contain starch and activity is stronger; and if the pollen grains are snuff colored, it is indicated that the pollen grains (FIG. 4) are hypogenetic. The experiment counts and analyses materials for 10 times repeatedly, namely 10 glass slides, one view is taken from each sheet, a standard deviation (See Table 1) of 10 times of a repeated average number is counted, and a diameter of the dyeing pollen is measured.

[0083] It is indicated from an experimental result that a probability of the rice event GSX2-55 dyed by the I.sub.2-KI is 51.68±2.89%, and far lower than the Hemeizhan (98.02±1.19%) and Huanghuazhan (98.28±1.47%). Theoretically, a transgenic element of the rice event GSX2-55 is positioned in a heterozygosis state in each generation, so a half of the pollen does not contain an exogenous gene (fertility), and a half of the pollen contains the transgenic element (sterility), ZmAA gene contained in the element is specific-expressed in the pollen, so starch in the pollen is effectively degraded, the pollen containing the transgenic element may not be dyed by the I.sub.2-KI, and pollen activity and fertility capacity are remarkably reduced, so transgenic pollen inactivation is caused.

TABLE-US-00001 TABLE 1 Pollen I.sub.2-KI dyeing experimental data statistics GSX2-55 Hemeizhan Dyeing Non-dyeing Dyeing Non-dyeing pollen pollen Dyeing pollen pollen Dyeing Plant number number number rate number number rate Repetition 1 66 58 53.23% 137 4 97.16% Repetition 2 76 70 52.05% 79 2 97.53% Repetition 3 92 66 58.23% 166 1 99.40% Repetition 4 76 73 51.01% 132 1 99.25% Repetition 5 98 101 49.25% 101 2 98.06% Repetition 6 95 101 48.47% 116 4 96.67% Repetition 7 68 73 48.23% 152 2 98.70% Repetition 8 131 114 53.47% 94 3 96.91% Repetition 9 76 77 49.67% 137 0 100.00% Repetition 10 92 81 53.18% 138 5 96.50% Ryeing rate 51.68 ± 2.89% 98.02 ± 1.19% average value Huanghuazhan Dyeing Non-dyeing pollen pollen Dyeing Plant number number number rate Repetition 1 114 0 100.00% Repetition 2 138 0 100.00% Repetition 3 135 1 99.26% Repetition 4 162 4 97.59% Repetition 5 180 3 98.36% Repetition 6 149 4 97.39% Repetition 7 153 0 100.00% Repetition 8 102 5 95.33% Repetition 9 130 3 97.74% Repetition 10 133 4 97.08% Ryeing rate 98.28 ± 1.47% average value

Embodiment 3: Statistic Analysis of Fluorescence Seed and Non-Fluorescence Seed Separation Proportion

[0084] DsRed gene may efficiently express red fluorescence protein in the seed of event GSX2-55, under excitation of green light, red fluorescence emitted by the DsRed protein may be used for distinguishing the rice event GSX2-55 and the rice recessive nucleus male sterile line Zhen18A seed (FIG. 3). The rice event GSX2-55 is selfing-fruited, after threshing, impurity removal and color sorting, red fluorescence seeds (GSX2-55) and non-fluorescence seeds (Zhen18A) in GSX2-55 selfing-fruited seeds are separated, and a grain number thereof is counted.

[0085] T.sub.4, T.sub.5 and T.sub.6-generation single plants (30 single plants are counted in each generation) of the rice event GSX2-55 obtained in the embodiment 1 are randomly selected, a separation rate of the harvested fluorescence seeds and non-fluorescence seeds thereof is calculated. A result is as shown in Table 2, a Chi-square X.sup.2 value (df=1) of the fluorescence separation proportion of all single plants is lower than a critical value 3.81, it is indicated that the separation rates of the fluorescence seeds and non-fluorescence seeds in T.sub.4, T.sub.5 and T.sub.6 three generations of the rice event GSX2-55 are in comply with 1:1.

TABLE-US-00002 TABLE 2 T.sub.4-T.sub.6 generation fluorescence separation rate data statistic analysis (Chi-square test) of the rice event GSX2-55 T4 T5 non- non- Fluorescence fluorescence fluorescence fluorescence Plant number seed seed X.sup.2.star-solid. seed seed X.sup.2.star-solid. Repetition 1 626 596 0.688 505 508 0.004 752 756 0.006 464 471 0.039 284 298 0.290 455 492 1.369 295 341 3.184 511 483 0.733 487 510 0.485 411 424 0.172 715 732 0.177 563 553 0.073 263 283 0.661 337 333 0.013 300 333 1.618 251 283 1.800 376 389 0.188 306 334 1.139 550 527 0.449 461 495 1.139 Repetition 2 530 576 1.831 265 306 2.802 518 485 1.021 397 419 0.540 444 469 0.631 423 436 0.168 185 185 0.003 341 354 0.207 310 355 2.911 330 347 0.378 365 399 1.425 511 482 0.790 285 276 0.114 432 468 1.361 416 390 0.775 383 373 0.107 442 484 1.815 431 426 0.019 365 373 0.066 532 510 0.423 Repetition 3 472 516 1.871 566 536 0.763 693 651 1.251 415 436 0.470 534 509 0.552 451 440 0.112 409 414 0.019 414 434 0.426 638 593 1.573 421 386 1.432 403 423 0.437 325 366 2.315 356 370 0.233 384 365 0.433 488 493 0.016 498 537 1.395 392 436 2.233 446 481 1.247 330 338 0.073 406 415 0.078 T6 non- Fluorescence fluorescence Plant number seed seed X.sup.2.star-solid. Repetition 1 1153 1172 0.130 932 947 0.110 1266 1261 0.006 800 826 0.373 1188 1243 1.196 1485 1466 0.111 1241 1243 0.000 1331 1300 0.334 1589 1619 0.262 1285 1215 1.932 Repetition 2 1532 1515 0.082 1144 1137 0.017 1368 1294 2.048 715 743 0.474 883 910 0.352 1043 1087 0.868 818 809 0.039 822 838 0.136 1079 1065 0.079 949 959 0.042 Repetition 3 481 519 1.369 735 759 0.354 474 497 0.498 405 426 0.481 605 666 2.832 707 733 0.434 558 558 0.001 673 733 2.476 568 623 2.448 896 860 0.698 Note: .sup..star-solid.represents that X.sup.2.sub.0.05 = 3.84 while df = 1.

[0086] It may be observed from the above embodiments 2 and 3 that the present disclosure realizes an disclosure purpose of stably creating rice male sterile line and maintainer line.

Embodiment 4: Flanking Sequence Analysis of the Rice Event GSX2-55

[0087] Through performing detailed analysis on the DNA inserted into rice genome containing event GSX2-55 and genome sequence of the insertion fragment flanking, molecular features of the rice event GSX2-55 are analyzed and known. These analysis includes: an insertion fragment sequence, an insertion number (a number of integration sites in rice genome), a copy number (copy number of the insertion fragments in the gene locus) of insertion fragments, integrity of an inserted gene cassette and a flanking sequence of the inserted fragment and the like.

[0088] Tail-POR and DNA sequencing technology is used for determining 5′ and 3′ junctions of the inserted fragment and the rice genome, and determining a complete DNA sequence (SEQ ID NO: 9) of the inserted fragment in the rice containing the event GSX2-55.

[0089] Flanking DNA sequence of the transgenic DNA sequence in the rice event GSX2-55 is cloned through HI-TAIL PCR (Liu et al., High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences (2007) BioTechniques 43:649-656) method. Operated in accordance with agricultural industry standards of the People's Republic of China NY/T674, the rice genomic DNA is extracted. The DNA is suitably diluted, an ultraviolet light absorption rate thereof in 260 nm and 280 nm is measured and recorded, and purified DNA concentration is calculated by an OD.sub.260 value which is equivalent to 50 μg/mL of the DNA concentration. According to the measured concentration, the DNA solution is diluted to 50 ng/μL, and stored in 4 DEG C. and used within a week. Those skilled in the art may improve this type of the method so as to extract the genomic DNA from any tissues of the rice. According to the HI-TAIL PCR method, a primer combination is designed, and the genomic DNA is used as a template, according to a primer length and a specificity difference, an asymmetric temperature cycle is designed, and the TAIL-PCR is performed, an obtained PCR product is separated and purified through agarose gel electrophoresis, after that, a standard DNA sequencing scheme is used for sequencing the DNA product. A sequencing result is as shown in (FIG. 1), 5′ flanking sequence in the left border flanking sequence of the transgenic DNA is as shown in SEQ ID NO: 7; 3′ flanking sequence in the right border flanking sequence of the transgenic DNA is as shown in SEQ ID NO: 8; and the transgenic DNA part completely integrated in the rice genomic DNA is as shown in SEQ ID NO: 9.

[0090] After the flanking sequence of the transgenic DNA of the rice event GSX2-55 acquired by the HI-TAIL PCR and DNA sequencing is contrasted and analyzed with indica rice 9311 genome database (http://www.gramene.org/,ASM465v1), it is discovered that the rice event GSX2-55 exogenous sequence is inserted at the long-arm terminal of a rice genome chromosome 12.

Embodiment 5: TAQMAN® Detection Method of the Rice Event GSX2-55

[0091] Extraction methods for genomes DNA of the rice event GSX2-55 and control samples (rice events FG2-47, FG2-205, FG2-525, FG2-670 and Zhen18A, corn Zheng 58, wheat ZhongguoChun, oilseed rape Zhongshuang 9 and soybean) are performed by referring to the embodiment 5. An ABI 7500FAST detection system is used for performing real-time PCR on each sample. A Taqman® probe TP55 (Applied Biosystems, Inc.) and primer combination TTY-55F/TTY-55R(SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13) is used for detecting the target sequence from the rice event GSX2-55. In addition, a second Taqman® probe and primer combination in allusion to rice endogenous sucrose phosphate synthase (SPS) gene is used for verifying that the amplified DNA (SEQ ID NO:15; SEQ ID NO: 16; SEQ ID NO:17; and SEQ ID NO: 18) exists in each real-time PCR reaction. The analysis verifies existence and/or inexistence of a real-time PCR qualitative positive and/or negative sample.

[0092] Positive and negative of the rice event GSX2-55 are determined on the basis of a Ct value of an event specificity target PCR, while a sample loading amount of the rice genomic DNA is 50 ng, an endogenous target and an event target are simultaneously amplified, and while the event target amplification Ct value is less than or equal to 36, a plant is assessed to be positive to the event; while the endogenous target and the event target are simultaneously amplified, and the event target amplification Ct value is greater than 36 and less than 40, the DNA is extracted again from the plant and real-time PCR analysis is repeatedly performed; if the endogenous target is amplified and the event target is not amplified (no amplification curve), the plant is assessed to be negative; if two targets are not amplified for a specific sample, it is determined that the sample is a DNA sample with poor quality or testing is failure, and the analysis is repeated.

[0093] It is shown from an experimental result that the rice event GSX2-55 is positive to an event specificity PCR result, but contrast rice (FG2-47, FG2-205, FG2-525, FG2-670 and Zhen18A), corn (Zheng 58), wheat (ZhongguoChun), oilseed rape (Zhongshuang 9) and soybean are negative to the event specificity PCR result (Table 3).

TABLE-US-00003 TABLE 3 specificity PCR analysis in the rice event GSX2-55 and contrast plants Rice endogenous Event specificity PCR SPS gene GSX2-55 + + FG2-47 − + FG2-205 − + FG2-525 − + FG2-670 − + Zhen18A − + Wuyun rice 7 − + Corn (Zheng 58) − − Oilseed rape − − (Zhongshuang 9) Wheat − − (ZhongguoChun) Soybean − − Note: event specificity PCR measuring summary of the rice event GSX2-55, herein the positive(+) indicates that the rice event GSX2-55 is existent; and the negative(−) indicates that the rice event GSX2-55 is not existent.

Embodiment 6: Sensitivity of TAQMAN® Detection Method of Rice Event GSX2-55

[0094] According to operating steps of plant genomic DNA extraction kit (DP350) of the TIANGEN BIOTECHBeijing Co., Ltd, genomic DNA of the rice event GSX2-55 and transformation acceptor Zhen18A is extracted, the genomic DNA of the rice event GSX2-55 is quantified to 10 ng/μL, 10 ng/μL of the Zhen18A genomic DNA is used as a diluent for continuously diluting the genomic DNA of the rice event GSX2-55 according to 10 times of a gradient (100%, 10%, 1%, 0.1%, 0.01%, 0.001%, 0.0001% and 0.00001%), and the diluent is used as a template for performing specificity PCR amplification (setting 3 parallels in each concentration gradient, and testing is repeated for 3 times), and used for detecting a lowest limit of detection (LOD) of the system (Table 4).

[0095] Through detecting the DNA of the rice event GSX2-55 in 10 times of the gradient, the quantitative PCR amplification system may effectively detect the DNA of the rice event GSX2-55 in different concentration, and shows a good linear relation (FIG. 2), a linear equation is Y=−3.25*Lg X+27.372; a coefficient of determination is R.sup.2=0.999; and quantitative PCR system amplification efficiency is 103.096%.

[0096] While a sample loading amount of the rice event GSX2-55 is 0.005 ng (0.01%), there is an apparent amplification curve, and a detection result may be judged to be positive. In other words, in a rice genomic DNA sample of which the sample loading amount is 50 ng, the quantitative PCR may specifically detect the DNA sample of which genomic DNA content of the rice event GSX2-55 is 0.01%.

TABLE-US-00004 TABLE 4 Standard curve Ct value of quantitative PCR system amplification GSX2-55 DNA sample loading amount (ng) Ct value 1 Ct value 2 Ct value 3 Ct mean 50 21.7481 21.77558 21.77645 21.76671 5 25.12457 25.09439 25.12196 25.11364 0.5 28.38204 28.35718 28.49834 28.41252 0.05 31.8074 31.75273 31.74871 31.76962 0.005 34.54358 34.92086 34.60136 34.6886